In article <simmenlab.33.2DF78727 at animal.ufl.edu>
simmenlab at animal.ufl.edu writes:
> Hello:
>> I am transfecting cells in culture with a b-gal expression
> vector. I was wondering if it was possible to detect b-gal expression without
> preparing cell extracts. I just want to know if the transfection worked or
> did not work. If anyone has any experience with this please let me know.
> Thank you very much!
>>> Karen Reed
For cultured endothelial cells I wash with PBS, fix with
2%Formaldehyde, 0.1%Glut, Wash again and stain O/N at 37C with X-Gal
assay buffer (60uls 50mg/ml X-Gal, 75ul each of K Ferro and
Ferricyanide (0.2M), 12uls MgCl2 (1M), made up to 3mls with PBS).
Worksd a treat. Can also use anti B gal antibodies (from lots of places
eg Sigma), or for in vivo work without fixation, Image green or Imagene
Red from Molecular Probes.
Good Luck
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Dave Bates, dobates at ucdavis.edu
Department of Human Physiology, University of California at Davis
No I don't have a sense of humour, now buy me a beer
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